Mdm2 and MdmX (also known as Mdm4) are key regulators of the p53 tumor suppressor. Previous work by our lab and by many other groups has determined that these MDM proteins complex with p53 and inhibit p53 functions, with Mdm2 inducing p53 ubiquitination and proteosomal degradation, and MdmX inhibiting p53 transactivation of p53 target gene expression. As such, Mdm2 and MdmX act as critical negative regulators of p53 tumor suppressing functions, and amplification of either the MDM2 or MDMX gene is observed in a significant percentage of human tumors. In this proposal, we summarize the progress we have made in our previous studies on p53 regulation by MDM and other cellular proteins. We also describe our very recent studies examining in vitro the effects of Mdm2 phosphorylation in the regulation of p53 stability, as well as our discovery of p53-independent roles for MdmX in suppressing proliferation and tumorigenesis in p53-null cells and mice. Based upon our preliminary data, we have generated new Mdm2 mouse models bearing amino acid substitutions in Mdm2 in order to examine in vivo the effects of Mdm2 phosphorylation on Mdm2-MdmX-p53 signaling and on p53 functions in development and tumor suppression. We also propose to further characterize p53-independent functions of MdmX in the regulation of cell transformation and chromosomal stability. Proposed research includes the generation of MdmX transgenic mice to model MdmX overexpression observed in human breast cancer, and to permit further exploration of the oncogenic and tumor suppressing capacity of MdmX in mammary tumorigenesis. This research should greatly increase our understanding of the roles of these MDM proteins in regulating cell growth and death, and the functional relationship between these homologues in DNA damage signaling and in p53-dependent and p53-independent suppression of cancer. PUBLIC HEALTH RELEVANCE: Mdm2 and MdmX (also known as Mdm4) are key negative regulators of the p53 tumor suppressor. Although much has been learned from transfection studies and from biochemical assays about the interconnecting roles of Mdm2 and MdmX in regulating p53, analysis of genetically modified mice has proven critical in substantiating the numerous and often conflicting models proposed for Mdm2 and MdmX regulation of p53 functions, and in developing new theories regarding the function of MDM proteins in cancer. We propose to analyze new mouse models to explore the effects of Mdm2 phosphorylation on Mdm2 and MdmX levels and on p53 functions in vivo. We also propose to continue our MdmX studies in cells and in mice to better understand and define the p53-independent roles for MdmX in transformation and chromosomal stability. We will also generate new MdmX transgenic models to explore p53- dependent and p53-independent effects of MdmX in mammary tumorigenesis. The results of these experiments will have clinical relevance, as an understanding of the regulatory mechanisms that control p53 activity in normal cell growth and in tumorigenesis is a critical prerequisite to developing new therapies to treat cancer in the significant percentage of patients who present with tumors bearing alterations in the Mdm2-MdmX-p53 signaling pathway.